Combined use of 13C- and 19F-NMR to analyse the mode of action andthe metabolism of the herbicide isoxaflutole

Serge Aubert*§, Kenneth E. Pallett

Plant Science Research Department, Rhône-Poulenc Agriculture Limited, Fyfield Road, Ongar, Essex, CM5 OHW, UnitedKingdom

* Author to whom correspondence should be addressed (fax +33 4 76 88 50 91; e-mail saubert@cea.fr)

(Received 30 November 1999; accepted 28 February 2000)

Abstract – 13C-Nuclear magnetic resonance was used to characterize the effects of the trifluoromethyl herbicide isoxaflutole onplant metabolism. A specific accumulation of tyrosine and phenylalanine in pea leaves was observed after treatment with thisherbicide, consistent with an inhibition of 4-hydroxyphenylpyruvate dioxygenase (HPPD), an enzyme involved in plastoquinoneand tocopherol synthesis and the known target of isoxaflutole.19F-NMR permitted the analysis of the uptake and degradationof isoxaflutole, with detection limits around 1 µM in perchloric acid extracts. Isoxaflutole was incorporated and transformed toan active diketonitrile derivative, which further underwent degradation to an inactive benzoic acid derivative. © 2000 Éditionsscientifiques et médicales Elsevier SAS

Aromatic amino acids / herbicide / isoxaflutole / metabolism / 13C-NMR / 19F-NMR

DKN, diketonitrile / HPPD, 4-hydroxyphenylpyruvate dioxygenase / TFA, trifluoroacetate

1. INTRODUCTION

Isoxaflutole (5-cyclopropylisoxazol-4-yl 2-mesyl-4-trifluromethylphenyl ketone) is a novel herbicide forpre-emergence control of many weeds causing bleach-ing of newly developed tissues in susceptible species.This symptom is typical of a broad range of herbicideswhich inhibit carotenoid synthesis. The majority ofthese inhibitors (such as diflufenican) targets phytoenedesaturase, the enzyme involved in the first twodesaturation steps in the conversion of the colourlesscarotenoid precursor phytoene into coloured caro-tenoids [18]. In contrast, it has been recently shownthat isoxaflutole belongs to a class of herbicides actingon 4-hydroxyphenylpyruvate dioxygenase (HPPD), anenzyme involved in the synthesis of plastoquinonesand tocopherols [16] (figure 1). Conventional14C-labelling studies have been used to identify this modeof action and analyse the metabolism of isoxaflutole.

NMR has been used in a few cases to analyseherbicide effects on plants (for a review, see [17]).

13C-NMR permits the detection of soluble metabolitesaccumulated in plant extracts at concentrations higherthan 1 µmol·g–1 w wt [2], thus allowing the character-ization of the effects of inhibitors on metabolism. Thistechnique has been used to analyse the effects ofglyphosate [9] and sulfonylureas [3] on heterotrophiccell metabolism.13C-NMR is usually not sensitiveenough to permit the analysis of the metabolism of theherbicide molecules. In the case of herbicides orpesticides containing phosphorus and applied at highconcentrations,31P-NMR can be used. This methodhas permitted the visualization and quantification insycamore cells of the herbicide glyphosate and itsmetabolite aminomethylphosphonate [9]. On the otherhand, many herbicides and other pesticides are fluori-nated molecules. This should allow the use of19F-NMR, which is much more sensitive than13C- and31P-NMR. 19F-NMR is widely used to follow themetabolic fate of fluorinated drugs in animals andhumans (for reviews, see [10, 12]).19F-NMR has alsobeen used for the detection of fluorinated pesticideresidues in food [14] or water [11]. More recently, themetabolic fate of a fluorinated fungicide [19] has beendescribed.

§ Present address: CEA-Grenoble, DBMS-PCV, 17, rue desMartyrs, 38054 Grenoble cedex 9, France.

Plant Physiol. Biochem., 2000,38 (6), 517−523 / © 2000 Éditions scientifiques et médicales Elsevier SAS. All rights reservedS0981942800007713/FLA

Plant Physiol. Biochem., 0981-9428/00/6/© 2000 E´ ditions scientifiques et médicales Elsevier SAS. All rights reserved

In this paper, we first describe the use of 13C-NMRto analyse the site of action and the metabolic effectsof the herbicide isoxaflutole, and second the use of19F-NMR to characterize its metabolic fate in plantcells.

2. RESULTS

2.1. Effects of isoxaflutole on heterotrophicmetabolism

Sycamore cells were incubated in the presence ofincreasing concentrations of isoxaflutole, in the range10 µM to 1 mM. 19F-NMR indicated that this herbi-cide was readily incorporated into cells (see below,next sections). However, no effect on growth rates andrespiration were observed. Similarly, 13C-NMR and31P-NMR on perchloric acid extracts failed to revealany significant effect of the herbicide treatment on thelevels of soluble metabolites, including sugars, aminoacids, organic acids, Pi, hexoses-P, nucleosides tri-phosphates (results not shown). This suggested thatisoxaflutole did not affect primary metabolism. Incontrast, and in agreement with reports in seedlings[16], sycamore cells exhibited severe bleaching, withcarotenoid contents reduced by 80 % after 2 weeks oftreatment (not shown). This indicated that isoxaflutolewas taken up by the cells and likely inhibited phytoenedesaturation. As the herbicide had no effect on theviability of the cells, we analysed the effect of isox-aflutole on autotrophic pea seedlings.

2.2. Effects of isoxaflutole on pea seedlingmetabolism

Pea seedlings were treated with isoxaflutole fromthe time of germination. They showed typical bleach-ing and growth arrest, preceding death (not shown, see[16]). In order to discriminate the specific effects ofisoxaflutole, we also treated pea seedlings withdiflufenican, an inhibitor of phytoene desaturase result-ing in symptoms similar to those of isoxaflutole [16].Perchloric extracts were prepared from aerial parts ofthe seedlings (shoot and leaves) and analysed using13C-NMR (figure 2). Glucose was the major sugar incontrol samples, while sucrose showed lower levels.As previously reported [6], homoserine (an intermedi-ate in the synthesis of aspartate-derived amino acids)was the major amino acid in pea seedlings, andglutamine, glutamate and asparagine were also detected(figure 2 A). Figure 2 B and table I show that isoxaflu-tole resulted in dramatic changes in the pattern ofamino acids. Homoserine became the major solute,and the amino acids glutamine and asparagine sharplyaccumulated. Other amino acids accumulated, includ-ing alanine, valine and lysine. However, similar effectswere observed in seedlings treated with diflufenican,indicating that they were related to bleaching. Incontrast, isoxaflutole treatment resulted in a markedaccumulation of tyrosine and phenylalanine (figure 3B, C and table I). Figure 3 shows the changes of thesearomatic amino acids over 9 d of treatment withisoxaflutole. Both accumulated at concentrations around

Figure 1. Schematic representation ofthe plastoquinone pathway, and its inte-gration in carotenoid biosynthetic path-way (from [15, 16, 20]). Solid arrowsrepresent the routes favoured by thebest evidence in most plant species.Enteric bacteria use the routes indicatedby dotted arrows. Inset (top right):structure of isoxaflutole (5-cyclo-propylisoxazol-4-yl 2-mesyl-4-trifluro-methylphenyl ketone). EPSP, 3-enol-pyruvylshikimate phosphate; PEP,phosphoenolpyruvate; HPPD, 4-hydro-xyphenylpyruvate dioxygenase; OHPP,4-hydroxyphenylpyruvate; PQ, plasto-quinone; PQH2, plastoquinol.

518 S. Aubert, K.E. Pallett

Plant Physiol. Biochem.

7–8 µmol·g–1 w wt after 9 d. These amino acids werenot detected (i.e. < 1 µmol·g–1 w wt, the detection limitof 13C-NMR with perchloric acid extracts) in controland diflufenican-treated seedlings.

2.3. Metabolic fate of isoxaflutole19F-NMR was used to characterize the metabolism

of isoxaflutole in sycamore cells. Perchloric acidextracts were prepared from cells incubated for vari-ous times with isoxaflutole (up to 7 weeks). Figure 4 Ashows that the herbicide was indeed taken up by the

cells, as suggested by the bleaching reported above.Isoxaflutole levels remained very low in cell extracts,but the spectra permitted to discriminate the quantifi-cation of two main products: diketonitrile (DKN) andbenzoate derivatives (see figure 4). DKN originatesfrom the cleavage of the isoxazole ring of the moleculeisoxaflutole, and has been shown to be the activecompound inhibiting HPPD [22]. We verified thatDKN resulted in similar effects as isoxaflutole onsycamore cells and pea seedlings (not shown). Abenzoate derivative originating from cleavage of DKNwas also detected in the extracts, and was inactive oncells and seedlings.

Figure 2. Representative 13C-NMR spectra obtained from perchloricacid extracts of pea seedlings (9 g wet weight of shoot + leaves). A,Control 9-d-old; B, seedlings treated with isoxaflutone for 9 d; C,seedlings treated with diflufenican for 9 d. Each spectrum (900 tran-sients) is representative of a perchloric acid extract obtained from 9 gfresh weight, and was recorded at 20 °C. Part of the spectra (15 to40 ppm) are enlarged (top left of each full spectrum). Peak assign-ments: Ala, alanine; Asn, asparagine; f, fructose; g, glucose; Gln,glutamine; Glu, glutamate; Hser, homoserine; Lys, lysine; Phe, phe-nylalanine; s, sucrose; Thr, threonine; Tyr, tyrosine; Val, valine.

Table I. Levels of various metabolites in control pea seedlings andpea seedlings treated with isoxaflutole or diflufenican for 7 d. Quan-tification was performed from 13C-NMR spectra (see Experimentalprocedures). Values are expressed as µmol·g–1 w wt and are the meanof five independent experiments (± SD).

Control Isoxaflutole Diflufenican(9 d) (9 d) (9 d)

Glucose 26.2 ± 4.2 18.5 ± 3.1 16.5 ± 2.1Sucrose 7.8 ± 1.2 7.5 ± 0.9 6.9 ± 1.3Alanine < 1 5.1 ± 1.2 3.5 ± 0.8Asparagine 6.5 ± 1.5 50.6 ± 6.5 12.7 ± 2.3Glutamate 5.4 ± 1.0 11.2 ± 1.6 7 ± 1.2Glutamine 2.1 ± 0.5 27.1 ± 3.2 37.2 ± 6.5Homoserine 6.1 ± 1.1 54.2 ± 8.5 39.4 ± 6.3Lysine < 1 4.3 ± 0.8 2 ± 0.3Phenylalanine < 1 7.1 ± 0.8 < 1Threonine < 1 4.5 ± 0.9 2.5 ± 0.5Tyrosine < 1 7.5 ± 1.2 < 1Valine < 1 4.0 ± 0.6 3 ± 0.7

Figure 3. Tyrosine and phenylalanine content in pea seedlings sub-jected to isoxaflutole treatment. Quantification were performed from13C-NMR spectra and are from representative experiments. Tyrosineand phenylalanine were not detected in control and diflufenican-treated pea seedlings.

NMR and the herbicide isoxaflutole 519

vol. 38 (6) 2000

The detection limit obtained using 19F-NMR wasaround 1 nmol·g–1 w wt and permitted the detection ofDKN in sycamore cells extracts after only 15 min of

exposure to isoxaflutole, indicating an efficient trans-port of the herbicide. Figure 4 B represents thetime-course evolution of DKN and benzoate in per-chloric acid extracts from sycamore cells during7 weeks of treatment. These molecules were quantifiedfrom the 19F-NMR spectra (see Methods). We observedthat DKN did not accumulate over time, and remainedroughly constant after the first 3 d. On the other hand,benzoate steadily accumulated, reflecting a constantrate of detoxification of DKN (around 0.5 nmol·d–1·g–1

w wt) during the first 2 weeks of treatment.

3. DISCUSSION

3.1. 13C-NMR analysis of the effects of herbicide

These results show that 13C-NMR permitted thecharacterization of the effects of isoxaflutole on plantmetabolism. This technique gives an overview of thepattern of major solutes in plant extracts (intracellularconcentration > 1 µmol·g–1 w wt), and therefore hasbeen used to characterize the effects of other herbi-cides on amino acid metabolism [3, 9]. Major changeswere observed in amino acid patterns after treatmentwith isoxaflutole. However, it was difficult to discrimi-nate the specific effects of isoxaflutole from theindirect effects due to rapid bleaching and arrest ofphotosynthesis. This was made possible by comparingthe effects of the inhibitor of phytoene desaturationdiflufenican, resulting in the same symptoms, but withdifferent metabolic repercussions.

On the one hand, we observed that both herbicidesresulted in an accumulation of various amino acids(see figure 2 and table I). These accumulations mayreflect the degradation of storage proteins, which arenot incorporated into newly synthesized proteins, dueto the arrest of photosynthesis and growth. In thiscontext, the huge accumulations of asparagine andglutamine may reflect high activity of ammoniumstorage. Asparagine has been characterized as a stressmolecule involved in ammonium storage during auto-phagic process [8] or other stresses including drought,increased salinity or mineral deficiency [21].Homoserine also markedly accumulated. Parentheti-cally, 13C-NMR confirmed the presence of high amountsof this amino acid in control plants, as previouslyreported by classic analytical method [6]. The role ofthis intermediate of aspartate-derived amino acids(methionine, threonine, leucine) remains to be eluci-dated.

On the other hand, we observed a marked accumu-lation of tyrosine and phenylalanine only in the plants

Figure 4. Use of 19F-NMR to analyse isoxaflutole degradation. A,Representative spectrum (16 384 transients) of perchloric acidextracts prepared from sycamore cells treated with 100 µM isoxaflu-tole for 3 d. The structures of isoxaflutole and its derivatives (DKNand benzoate) are represented. The star on the structure of DKN showsthe asymmetric carbon responsible for the presence of two isoforms ofthe compound. DKN, diketonitrile; TFA, trifluoroacetate. B, Forma-tion of DKN and benzoate derivatives in sycamore cells treated with100 µM isoxaflutole. Quantifications were carried out according to theprocedure described in Methods. Standard deviations (< 10 %) are notrepresented for clarity.

520 S. Aubert, K.E. Pallett

Plant Physiol. Biochem.

treated with isoxaflutole. This suggested that theseaccumulations were specific to this treatment. This isconsistent with an inhibition of HPPD by isoxaflutole,as characterized in vitro [22]. Interestingly, the firstclue to the mechanism of action of HPPD inhibitorshas been the discovery that treated rats were found tobe tyrosinemic. 13C-NMR analysis would have permit-ted an easy evidence of tyrosine accumulation from anunpurified leaf extract, thus orientating the research ofthe target site of the new herbicide. The accumulationof tyrosine probably corresponds to an inhibition of itscatabolism, which has been suggested to involveHPPD [13]. Concerning phenylalanine, little is knownabout its pathways of catabolism [20]. An enzymaticactivity catalysing the conversion of phenylalanine totyrosine has been reported [13]. However, furtherinvestigations will be necessary to document thecross-regulations presumably involved in aromaticamino acid metabolism and to understand phenylala-nine accumulation in isoxaflutole-treated plants.

The symptomology of bleaching after isoxaflutoletreatment is most probably explained by the arrest ofplastoquinone synthesis, since these compounds origi-nate from homogentisate, the product of HPPD (seeschematic in figure 1). Indeed, it has been recentlydemonstrated that plastoquinones are an essential com-ponent of phytoene desaturation [15]. No effects onheterotrophic sycamore cells were observed in termsof respiration, growth rates or metabolite pattern,while carotenoids were reduced to less than 10 % oftheir normal content in these cells after 2 weeks. Thissuggested that carotenoids are not necessary for growthof these heterotrophic cells under normal conditions.However, preliminary results suggest that carotenoid-depressed cells were much less resistant to activeoxygen radicals (not shown), consistent with the roleof carotenoid pigments as scavengers [1].

3.2. 19F-NMR analysis of herbicide metabolicfate

Suspension cells represent an attractive model toinvestigate herbicide metabolism [3, 9, 17]. Our resultsshow, for the first time, that 19F-NMR can be used asa relatively sensitive tool to analyse fluorinated herbi-cide molecules. The technique presents the followingadvantages. First, detection limits around 1 nmol·g–1

w wt were obtained, which is three orders of magni-tude lower than 13C-NMR detection limit. Second, thecompounds can be easily detected in complex plantextracts without requiring difficult and time-consumingpurification steps. Indeed, plant extracts do not syn-thesize fluorinated compounds (except some rare plants,

see [4]). In addition, the fluorine chemical shift ishighly sensitive, permitting keen separations and detec-tion of changes far away from the fluorine atoms (seefigure 4 A). Third, it avoids the use of radioactive14C-compounds, attaining comparative detection lim-its (see [19] for the case of a fungicide).

This analysis shows the extremely rapid penetrationof isoxaflutole in suspension cells (after 15 min oftreatment). This is presumably different in leaves,where various barriers must be overcome (cuticule,epidermis). We could also quantify the rate of herbi-cide metabolism (around 0.5 nmol·d–1·g–1 w wt). Thisrelatively rapid and simple pathway of isoxaflutoledegradation in suspension cells is identical to that seenin whole plants using radiolabelled studies [16].

4. METHODS

4.1. Chemicals

Isoxaflutole, its diketonitrile derivative (DKN), andbenzoic acid derivatives were synthesized in the chemi-cal laboratories of Rhône-PoulencAgriculture ResearchDivision. Diflufenican was also obtained from thesame source.

4.2. Materials

Cell suspensions were chosen in order to improvethe homogeneity of the incubation conditions andfacilitate herbicide uptake. Sycamore (Acer pseudo-platanus L.) cells used in the present study were grownat 20 °C as a suspension in liquid nutrient mediaaccording to Bligny and Leguay [5]. The culturemedium was kept at a volume of 0.3 L and stirredcontinuously at 60 rpm. Under these conditions, thecell number doubling time was 40–48 h after a lagphase of approx. 2 d and the maximum cell densitywas attained after 7–8 d of growth, when the stationaryphase is attained. The cell suspensions were main-tained in exponential growth by sub-culturing every7 d. The cell wet weight was measured after strainingculture aliquots onto a glass-fibre filter. Pea seeds(Pisum sativum L. var. Douce Provence) were grownfrom seeds in vermiculite. Once emerged, seedlingswere sprayed with water containing 0.5 % Tween 20and 50 µM isoxaflutole or 50 µM diflufenican, using astandard greenhouse sprayer fitted with a fan-jet nozzle.The plants were then grown on a 12-h light (26 °C),12-h dark (20 °C) cycle. After various times, shoot andleaves were excised and immediately frozen in liquidnitrogen for perchloric acid extraction.

NMR and the herbicide isoxaflutole 521

vol. 38 (6) 2000

4.3. Carotenoid determinationTotal carotenoid content in aerial parts of pea

seedlings were determined according to Davies [7].

4.4. NMR analysis

4.4.1. Perchloric acid extract preparation

For perchloric acid extraction, cells or seedlings(9 g wet weight) were quickly frozen in liquid nitrogenand ground to a fine powder with a mortar and pestlewith 1 mL 70 % (v/v) perchloric acid. The frozenpowder was then placed at –10 °C and thawed. Thethick suspension thus obtained was centrifuged at15 000 × g for 10 min to remove particulate matter,and the supernatant was neutralized with 2 M KHCO3

to about pH 5. The supernatant was then centrifuged at10 000 × g for 10 min to remove KClO4; the resultingsupernatant was lyophilized and stored in liquid nitro-gen. For 13C-NMR analysis, this freeze-dried materialwas redissolved in 2.5 mL water containing 10 %D2O, neutralized to pH 7.5. For 19F-NMR analysis, thefreeze-dried material was redissolved in 0.5 mL watercontaining 50 % D2O and 50 µM TFA (trifluoroaceticacid) as reference, and neutralized to pH 7.5.

4.4.2. NMR measurements

4.4.2.1. 13C-NMR analysisSpectra of neutralized perchloric acid extracts were

recorded on a Bruker NMR spectrometer (AMX 400,WB) equipped with a 10-mm multinuclear probe tunedat 100.6 MHz. The deuterium resonance of D2O wasused as a lock signal. Acquisition conditions used are:90° radio frequency pulses (19 µs) at 6-s intervals;spectral width 20 000 Hz; 900 scans; Waltz-16 1Hdecoupling sequence (with two levels of decoupling:2.5 W during acquisition time, 0.5 W during delay).Free induction decays were collected as 16K datapoints, zero filled to 32K and processed with a 0.2-Hzexponential line broadening. Spectra are referenced tohexamethyldisiloxane at 2.7 ppm.

4.4.2.2. 19F-NMR analysisSpectra of neutralized perchloric acid extracts were

recorded on a Bruker NMR spectrometer (AH 400,NB) equipped with a 5-mm 19F/1H probe tuned at376.5 MHz. Spectra are referenced to trifluoroacetateat –76.5 ppm.

4.4.3. Identification and quantification

4.4.3.1. 13C-NMR analysisSpectra of standard solutions of known compounds

at pH 7.5 were compared with that of a perchloric acid

extract of sycamore cells. The definitive assignmentswere made after running a series of spectra obtainedby addition of the authentic compounds to the perchlo-ric acid extracts, according to previous publications(see [2]). To determine accurately the total amounts ofamino acids in perchloric extracts, we proceeded asfollows: a 20-s recycling time was used to obtain fullyrelaxed spectra, and the calibration of the peak inten-sities by the addition of known amounts of thecorresponding authentic compounds. The possibleerrors in the measurements, as a consequence ofperchloric acid extraction, were estimated by addingknown amounts of authentic compounds to frozencells before grinding. We have observed that, for allthe compounds studied, the overall yield of recoverywas ≈80 %.

4.4.3.2. 19F-NMR analysisThe compounds were identified and quantified by

addition of the isoxaflutole derivatives synthesized byRhône-Poulenc Agriculture.

Acknowledgments

This work has been conducted under the BIOAVE-NIR Program, funded by Rhône-Poulenc with contri-bution of the ‘Ministère de la Recherche et de l’Espace’and the ‘Ministère de l’ Industrie et du CommerceExtérieur’ . The support of the ‘ laboratoire de réso-nance magnétique en biologie et métabolisme’ isacknowledged.

REFERENCES

[1] Asada K., Mechanisms for scavenging reactive mol-ecules generated in chloroplasts under light stress, in:Baker N.R., Bowyer J.R. (Eds.), Photoinhibition ofPhotosynthesis, Bios Scientific Publishers, Oxford,1994, pp. 129–142.

[2] Aubert S., Bligny R., Douce R., NMR studies ofmetabolism in cell suspensions and tissue cultures, in:Shachar-Hill Y., Pfeffer P. (Eds.), Nuclear MagneticResonance in Plant Biology, American Society of PlantPhysiologists, Rockville, USA, 1996, pp. 109–154.

[3] Aubert S., Day D.A., Whelan J., Bligny R., Douce R.,Induction of alternative oxidase synthesis by herbi-cides inhibiting branched-chain amino acid synthe-sis, Plant J. 11 (1997) 649–657.

522 S. Aubert, K.E. Pallett

Plant Physiol. Biochem.

[4] Baron M.L., Bothroyd C.M., Rogers G.I., Staffa A.,Rae I.D., Detection and measurement of fluoroacetatein plant extracts by 19F NMR, Phytochemistry 26(1987) 2293–2295.

[5] Bligny R., Leguay J.J., Techniques of cell suspensionculture, Methods Enzymol. 148 (1987) 3–16.

[6] Bryan J.K., Synthesis of the aspartate family andbranched-chain amino acids, in: Stumpf P.K., ConnE.E. (Eds.), The Biochemistry of Plants, vol. 5, Aca-demic Press, New York, 1980, pp. 403–453.

[7] Davies B.H., Carotenoids, in: Goodwin T.W. (Ed.),Chemistry and Biochemistry of Plant Pigments, Aca-demic Press, New York, 1976, pp. 91–162.

[8] Génix P., Bligny R., Martin J.B., Douce R., Transientaccumulation of asparagine in sycamore cells after along period of sucrose starvation, Plant Physiol. 94(1990) 717–722.

[9] Gout E., Bligny R., Génix P., Tissut M., Douce R.,Effect of glyphosate on plant cell metabolism. 31P and13C NMR studies, Biochimie 74 (1992) 875–882.

[10] Komorosky R.A., In vivo NMR of drugs, J. Am.Chem. Soc. 66 (1994) 1024–1033.

[11] Mabury S.A., Crosby D.G., 19F-NMR as an analyticaltool for fluorinated agrochemical research, J. Agric.Food Chem. 43 (1995) 1845–1848.

[12] Malet-Martino M.C., Martino R., Magnetic resonancespectroscopy: a powerful tool for drug metabolismstudies, Biochimie 74 (1992) 785–800.

[13] Mazelis M., Amino acid catabolism, in: Stumpf P.K.,Conn E.E. (Eds.), The Biochemistry of Plants, vol. 5,Academic Press, New York, 1980, pp. 541–567.

[14] Mortimer R.D., Black D.B., Dawson B.A., Pesticideresidue analysis in foods by NMR. 3. Combination of19F-NMR and GC-ECD for analyzing trifluraline resi-dues in field-grown carrots, J. Agric. Food Chem. 42(1994) 1713–1716.

[15] Norris S.R., Barrette T.R., DellaPenna D., Geneticdissection of carotenoid synthesis in Arabidopsis definesplastoquinone as an essential component of phytoenedesaturation, Plant Cell 7 (1995) 2139–2149.

[16] Pallett K.E., Little J.P., Sheekey M., Veerasekaran P.,The mode of action of isoxaflutole. I. Physiologicaleffects, metabolism, and selectivity, Pestic. Biochem.Physiol. 62 (1998) 113–124.

[17] Ratcliffe R.G., Roscher A., Prospects for in vivo NMRmethods in xenobiotic research in plants, Biodegrada-tion 9 (1999) 411–422.

[18] Sandmann G., Böger P., Inhibition of carotenoid bio-synthesis by herbicides, in: Böger P., Sandmann G.(Eds.), Target Sites of Herbicide Action, CRC Press,Boca Raton, FL, 1989, pp. 25–38.

[19] Serre A.M., Roby C., Roscher A., Nurit F., Euvrard M.,Tissut M., Comparative detection of fluorinated xeno-biotics and their metabolites through 19F NMR or 14Clabel in plant cells, J. Agric. Food Chem. 45 (1997)242–248.

[20] Siehl D.L., The biosynthesis of tryptophan, tyrosine,and phenylalanine from chorismate, in: Singh B.K.(Ed.), Plant Amino Acids, Biochemistry andBiotechnology, Marcel Dekker Inc., New York,1998, pp. 171–204.

[21] Stewart G.R., Lahrer F., Accumulation of amino acidsand related compounds in relation to environmentalstress, in: Miflin B.J. (Ed.), Amino Acids andDerivatives, Academic Press, New York,1980, pp. 609–635.

[22] Viviani F., Little J.P., Pallett K.E., The mode of actionof isoxaflutole. II. Characterization of the inhibition ofcarrot 4-hydroxyphenylpyruvate dioxygenase by thediketonitrile derivative of isoxaflutole, Pestic. Bio-chem. Physiol. 62 (1998) 125–134.

NMR and the herbicide isoxaflutole 523

vol. 38 (6) 2000